Table 1 shows the impurity levels for various Pfizer antibodies using the processes outlined in Figure 1. The impurity profiles
are collected after the final chromatography step. The percent monomer is determined by size exclusion high performance liquid
chromatography, using two G3000 SWXL columns (Tosoh, Montgomeryville, PA) in series with detection of the antibody at 214
nm. HCP is detected using a high-sensitivity sandwich Enzyme-Linked ImmunoSorbent Assay (ELISA) kit specific for either CHO
or NS0 proteins following the manufacturer's instructions (Cygnus Technologies, Southport, NC). We measured residual recombinant
protein A levels using a sandwich ELISA kit from Repligen (Waltham, MA). Finally, residual total DNA was evaluated by extraction
using the DNA extractor kit from Wako (Richmond, VA), followed by detection of the single-stranded DNA using a threshold kit
(Molecular Devices, Sunnyvale, CA).
Table 1. Impurity profiles and classifications for various therapeutic monoclonal antibodies using the platform processes
outlined in this article
The results indicate that for the representative antibodies shown for the different process permutations, the current purification
scheme as outlined is sufficient to produce an acceptable product. A few of the antibodies required minor changes to the platform
process to produce the desired product purity, as explained in Table 1. Figure 3 shows the overall virus reduction achieved
for the early-phase representative antibodies in the different platform processes. Again, the results indicate that all processes
achieved satisfactory viral clearance. However, the current process is advantageous as we increase column capacities to accommodate
increasing titers, as insurance for acceptable virus clearance results.
Figure 3. Graph of the log viral clearance values obtained for the therapeutic antibodies shown in Table 1. The changes in
color correspond to the processes outlined in Figure 1. From left to right: original process, process 2, process 3, and current
The current process, as shown in Figure 1, provides Pfizer with a combination of purification and virus clearance robustness,
allowing us to quickly move products into the pilot plant for clinical manufacturing, thus accelerating progress toward the
clinic. Even though the other processes shown in Figure 1 also met these criteria, they did not meet our new standard for
robustness established by increasing product titers. A process was needed that was more adept at handling changes in the composition
of the cell culture broth as well as unexpected increases in titer following scale-up, requiring additional column capacities.
Although the current process bucks the trend of reducing the number of unit operations, it increases the chance of meeting
the purification needs of a greater number of projects in a shorter time frame. These early-phase purification processes can
be optimized further once proof of concept has been achieved.
As cell culture titers continue to increase, the biopharmaceuticals industry will be faced with new challenges, including
greater product heterogeneity and increasing impurity levels. As scale increases for early-phase manufacturing, resin capacity
must increase to minimize operating costs, and therefore, it will be necessary to carry out studies to determine the impact
of these changes on virus clearance and the removal of impurities. If increased capacity cannot meet these needs, alternative
separation methods such as simulated moving bed chromatography will become paramount.
It also will become necessary to increase the throughput of nanofiltration devices, so that they do not become the next process
bottleneck. In addition, we must develop new technologies for ultrafiltration to meet the need for high-concentration drugs
used for subcutaneous injection. These are just some of the challenges facing the industry today.